CN104736167A - Lentiviral vectors containing an mhc class i promoter - Google Patents
Lentiviral vectors containing an mhc class i promoter Download PDFInfo
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- CN104736167A CN104736167A CN201380026705.4A CN201380026705A CN104736167A CN 104736167 A CN104736167 A CN 104736167A CN 201380026705 A CN201380026705 A CN 201380026705A CN 104736167 A CN104736167 A CN 104736167A
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Abstract
The present invention relates to the insertion of a promoter sequence from an MHC class I gene promoter into a lentiviral vector in order to direct the transcription of a transgene, which preferably encodes an immunogenic polypeptide to be expressed in a mammalian cell host, preferably APC (DCs). The invention encompasses these vectors, methods of making the vectors, and methods of using them, including medicinal uses.
Description
Technical field
The domain variability that the invention belongs to recombiant vaccine technology relates to the improvement of slow virus carrier, and it can be used as therapeutic and preventative vaccine.The carrier comprising MHC I type (MHCI) promoter provides the characteristic of the improvement being better than other carriers.
Background
Recombiant vaccine develops with the progress of recombinant DNA technology, allows modification virus genome to produce the virus of modifying.In this way, genetic sequence can be introduced non-pathogenic virus, the immunogenic protein for the treatment of to express in target cell after infection thus it is encoded, to develop specific immunne response in its host.
This type of vaccine constitutes the major progress (Kutzler etc., Nat Rev Genet, 9 (10): 776-788,2008) in vaccine technologies.Specifically, it has the advantage being better than traditional vaccine: avoid living (attenuation) virus and eliminating the risk be associated with inactivated vaccine manufacture.
The gene delivery of modified retrovirus (retroviral vector) is adopted to be described (Cell, 33 (1): 153-9,1983) by Mann etc. in early days in the eighties in last century.The most frequently used oncogenicity retrovirus is based on Moloney (Moloney) murine leukemia virus (MLV).Its genome is simple, and polyprotein Gag, Pol and Env produce from this genome and need to carry out virus replication (Breckpot etc., 2007, Gene Ther, 14 (11): 847-62 with trans forms; He etc., 2007, Expert Rev vaccines, 6 (6): 913-24).The sequence generally needed with cis is long terminal repeat (LTR) and following peripheral sequence thereof: the inverted repeat (IR or att site) needed for integration, packaging sequence Ψ, transfer RNA binding site (primer binding site, and relate to some other sequences (the repetition R in the LTR that positive start of chain is required and polypurine sequence (PPT)) of reverse transcription PBS).In order to generate Replication defective retroviral vector, usually make gag, pol and env gene all lack, and substitute with expression cassette.
The retroviral vector (i.e. slow virus carrier) being derived from lentiviral gene group has emerged in large numbers for the instrument likely for gene therapy and immunization therapy object, because their displays are better than some advantages of other viral system.Specifically, slow virus carrier itself is nontoxic, and unlike other retrovirus, slow virus can transduce non-dividing cells, especially dendritic cell (He etc., 2007, Expert Rev vaccines, 6 (6): 913-24), this allows the antigen presentation by endogenous path.
Slow virus is by genetic constitution similarity, the molecular mechanism copied and be connected with the biological interaction of its host.The most it is well known that it is the syndromic cause of disease of slow disease that is initial and slowly development of hiding after long-term subclinical infection; Therefore, virus (Narayan etc., 1989, J Gen Virol, 70 (7): 1617-39) that it is called as " slowly ".Its basic comprising is identical with retrovirus used but more complicated due to the existence of subsidiary gene (such as vif, vpr, vpu, nef, tat and rev), and described subsidiary gene plays a crucial role in copying in the body of slow virus.
Slow virus represents a lentivirus of Retroviridae (Retroviridae), and it comprises human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious encephalitis (EIAV), Caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV) and feline immunodeficiency virus (FIV).Slow virus can ad infinitum be continuously present in its host, and with friction speed continuous replication between the infection period in all one's life.Virus continuing in its host is copied and depends on that it avoids the ability of host defense.
The design of recombination and integration type slow virus carrier is based on the cis of slow virus and being separated of transacting sequence.Effective transduction in Unseparated Cell needs to there are two kinds of cis acting sequences in lentiviral gene group: center polypurine sequence (cPPT) and central termination sequence (CTS).This causes formation to be called the three stranded DNA structure of center DNA " lobe (flap) ", this structure makes gene enter maximizing efficiency (Zennou etc. in Unseparated Cell (comprising dendritic cell (DC)) core, 2000, Cell, 101 (2) 173-85; Arhel etc., 2007, EMBOJ, 26 (12): 3025-37).
Dendritic cell have important function for antigen presentation, because which constitute with present antigen and initial vaccination response as the main Types of the antigen presenting cell (APC) of major function.
For producing immunne response, by cell, antigen protein must be processed into peptide, described peptide is shown at cell surface by MHC albumen (MHC).Circulation APC in draining lymph node by peptide-MHC complex submission to T cell, it amounts to activated T cell together with stimulus signal with φt cell receptor interaction parallel connection herein.
Multinomial research shows, uses the inoculation of slow virus carrier to cause DC to carry out antigen presentation and Antigen-specific cytotoxic T lymphocyte (CTL; CD8
+t cell) strong activation.Therefore, in the past 10 years, slow virus carrier is carried out engineered for gene transfer and immunization therapy application.
By removing LTR U3 sequence, producing " automatic inactivation " carrier not containing viral promotors and the enhancer sequence originally existed in LTR completely, thus having improved the safety of slow virus carrier.
By recombinant technique, after using different DNA plasmids to HEK 293T people cultured cell transient transfection, produce the lentiviral particle containing slow virus carrier, described plasmid comprises:
(i) packaging plasmid, it expresses at least Gag, PolRev, Tat, and expresses the necessary structure of packaging and the pheron of transfer construct in some cases;
(ii) transferring plasmid, it contains the necessary expression cassette of packaging, reverse transcription and integration and HIV cis-acting factors; And
(iii) plasmid of coding peplos, the glycoprotein of vesicular stomatitis virus (VSV.G) as a rule, this albumen allows formation can the hybrid particles (false type) of targeting various kinds of cell (especially ajor histocompatibility (MHC) antigen presenting cell (APC), comprises DC).
The method can make the cell of transfection instantaneous production lentiviral particle carrier.But, also by inserting cellular genome by stable to packaging gene, provirus coding DNA and env gene, make cell production lentiviral particle constantly carrier.This allows under the condition not needing transient infections by cell production lentiviral particle constantly carrier.Certainly, also can use the combination of these methods, wherein by some DNA/ plasmid integrations to cellular genome other provided by transient transfection.
Circles slow virus carrier is just being designed to reduce the risk occurred with the latent carcinoma that is associated of insertion mutagenic event, is used in particular for vaccination object:
In the vaccination based on the integrated slow virus carrier of direct injection antigen encoding, the cell of expressing the transduction of related antigen becomes the target of the immunne response of initiation and removes from vaccinated organism within a couple of days or several weeks.
In addition, in the slow virus carrier of automatic inactivation, the disappearance in the U3 region of the 3'LTR of viral promotors and enhancer sequence limits the probability that internal promoter activates.This disappearance directly represent the experience that SCID-X1 gene therapy experiments that 1998-1999 carries out obtains, this test uses the retroviral vector based on Moroni virus to carry out (Cavazzana-Calvo etc. for the child of the X suffering from rare type chain (SCID-X1 gene) severe immune deficiency disease, 2000, Science., 288 (5466): 669-72).This duration of test, the retrovirus in source, Moroni causes 4 in 9 children to develop leukemia (Hacein-Bey-Abina etc. in the integration very near people LM02 proto-oncogene place, 2008, J.Clin.Invest., 118 (9): 3132-3142).This shows, malignant tumor is the result of viral U3 promoter/enhancer close to LM02 proto-oncogene.
Enhancer is cis acting sequence, and it can separated by a distancely work as activating transcription factor.It has been widely used in the carrier of viral source, because it seems with regard to the most effective (Chinnasamy with regard to various kinds of cell type (particularly DC) middle acquisition transgenic strongly expressed, Chinnasamy etc., 2000, HumGene Ther 11 (13): 1901-9; Rouas etc., 2008, Cancer Gene Ther 9 (9): 715-24; Kimura etc., 2007, Mol Ther 15 (7): 1390-9; Gruh etc., 2008, J Gene Med 10 (1) 21-32).But, consider and insert the safety problem of mutation, this kind of transcription enhancer sequences should be deleted from slow virus carrier construct to destroy the insertion mutation risk that enhancer kindred effect produces.Up to now, this enhancer kindred effect is modal insertion mutation mechanism and is the unique effect described in the case of gene transfer descendant or animal tumor generation event.
Therefore, need research and development not comprise the retrovirus, particularly slow virus carrier of virus enhancer, it allows the enough expression of immunogenic peptide of transgenes encoding, and if possible, that observes when expression and use CMV promoter is suitable.
A nearest research reports and uses MHC II type gene (MHC II type) (Kimura etc., 2007, MolTher 15 (7): 1390-9) and dectin-2 gene (Lopes etc., 2008, JVirol 82 (1): 86-95) the DC specificity promoter of originating replaces viral promotors.The dectin-2 gene promoter that Lopes etc. use comprises enhancer and the adenovirus conserved sequence (oppositely terminal repetition in adenovirus promoter) (Bonkabara etc., 2001, J.Immunology, 167:6893-6900) of presumption.The MHC II type gene promoter that Kimura etc. use is not containing any known enhancer.
But, when there is no enhancer, find that MHC II type promoter cannot provide enough transgene expressions in DC.Specifically, contrary with the viewed immunne response of use CMV promoter/enhancer, the slow virus carrier comprising MHC II type promoter fails to cause immunoreation in immunocompetence C57BL/6 mice.Although observed the transgene expression integrated and continue in mice after injection, cannot be replied by stimulator antigen specific C D8+ cytotoxic T lymphocyte by the slow virus carrier of MHC II type promoter transcription, be also even like this after vaccination is strengthened.Therefore authors of these researchs sum up, and the employing of MHC II type promoter is only for needing the application of continuous expression to be interesting in gene replacement therapies, but really not so in immunization therapy.
Therefore, reply for for antigen induced immune, MHC II type promoter is not suitable slow virus carrier promoter.In addition, dectin-2 promoter has dendritic cell specificity, and it cannot eliminate the carrier be integrated in other non-expressing cell types.And dectin-2 promoter seems containing enhancer.Therefore, due to security reason, dectin-2 promoter is not good slow virus carrier promoter.
Preferably, in immunization therapy, slow virus carrier provides effective transgene expression, its cause needed for specific immune response.This needs the expression in APC (as dendritic cell) to be in high level.
Also preferably by the immunne response removing slow virus carrier cell of transduceing to provide higher levels of safety.That is, the immunne response produced for transgenic can cause the immunne response being enough to remove slow virus carrier transducer cell in host.The removing of transducer cell eliminates the persistence of slow virus carrier in host and the second order effect that this carrier is possible.For removing transducer cell, the expression in non-dendritic cell needs to have the level allowing to be eliminated by immunne response.
Meanwhile, promoter should by originally making immunostimulation maximization with the key cells (i.e. dendritic cell) that relates in the activation of memory T cell and should making to cause the risk minimization of insertion mutation in the stem cell of malignant tumor and genotoxicity.Therefore, promoter should have sufficiently high activity in dendron and other cells, but not containing enhancer.Based on these standards, due to the existence of strong enhancer, viral promotors (as CMV promoter) is not desirable.These standards are summarized as follows:
1. in the antigen presenting cell (comprising dendritic cell) high expressed to induce maximum immunne response;
2. other transduction cell types in enough horizontal expressions, this expression be enough to for by induce immunne response eliminate; And
3. lack enhancer element to avoid inserting effect.
Therefore, there are the needs for improved carrier in this area.Present invention accomplishes these needs in this area.
Summary of the invention
The present invention includes the compositions comprising slow virus carrier and the method using described carrier.In one embodiment, the present invention includes a kind of slow virus carrier, it comprises the transgenic sequence of encoding immunogenic polypeptide, and this transgenic sequence transcribes control by MHC I type promoter; Compared with the carrier of transcribing control being subject to CMV promoter with transgenic sequence, the immunogenic polypeptide of this carrier pin to coding induces stronger CTL response in vivo.
Preferably, MHC I type promoter is HLA-A2 promoter, HLA-B7 promoter, HLA-Cw5 promoter, HLA-E promoter or HLA-F promoter.
In multiple embodiment, promoter sequence comprise have more than 90% with promoter sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4 or SEQ ID NO:5, polynucleotide sequence preferably greater than 95%, more preferably above 99% homogeny.
In multiple embodiment, carrier comprises promoter and the LTR of enhancer disappearance, cPPT/CTS sequence, Ψ (psi) sequence and/or two the slow virus LTR from slow virus of U3.
In a preferred embodiment, slow virus carrier is not containing enhancer.
The immunogenic polypeptide of transgenes encoding comprises the antigen of Gag, Pol and/or Nef albumen from HIV virus.This antigen can be at least one tumor antigen.
The present invention also comprises the host cell of the separation comprising slow virus carrier of the present invention.
The present invention also comprises the method for the production of slow virus carrier granule.These class methods can comprise the following steps: use the host cell that slow virus carrier transfection is suitable; Use the packaging plasmid carrier transfection host cell containing viral DNA sequences, the retroviral at least structure of this DNA sequence encoding and integrase protein; Cultivate the host cell of described transfection to make described slow virus carrier express and to be packaged into slow virus carrier granule; And results are expressed and pack the slow virus carrier granule formed in the host cell cultivated.
The present invention also comprises the slow virus carrier granule comprising slow virus carrier of the present invention.Slow virus carrier granule can comprise cPPT/CTS polynucleotide sequence; The LTR of the promoter of U3 and enhancer disappearance; And/or the transgenic sequence under the control of MHCI type promoter.Preferably, promoter sequence comprise have more than 90% with promoter sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, polynucleotide sequence preferably greater than 95%, more preferably above 99% homogeny.
The present invention also comprises the compositions comprising slow virus carrier of the present invention or lentiviral particle, as medicine or vaccine.
The present invention also comprises the method for inducing T cell response, and comprise and give patient's slow virus carrier granule, described slow virus carrier granule comprises cPPT/CTS polynucleotide sequence; The LTR of the promoter of U3 and enhancer disappearance; And/or the transgenic sequence under the control of MHC I type promoter.
Brief Description Of Drawings
The schematic diagram of Fig. 1 .MHCI and MCHII promoter.
The controlling element that the display of MHC I type (MHCI) promoter is conservative: κ B (NF-κ B binding site), ISRE (IFN-stimulated response element), SXY (SXY controlling element), TATA (transcription signal) and ATG (start codon for transgenic transduction).MCHII promoter family only shows SXY, TATA and ATG controlling element.
Fig. 2. in two kinds of different cell types, the GFP of multiple promoters driven expresses.
Use has the slow virus carrier construct of the multiple promoteres be connected with green fluorescent protein (GFP) gene to HEK293T and BDCM cell of transduceing, and assesses the ability of promoters driven GFP expression.
The induction that Fig. 3 A and B. many kinds of interferon are expressed GDP
Deposit in case at multiple interferon molecule, use the slow virus carrier with multiple promoter to HEK293T and BDCM cell of transduceing.With the ability that later evaluation promoters driven GFP expresses.Show the MFI in 293T cell (A) and BDCM cell (B).Be also tested for MHCI and the β 2m promoter weakening form, wherein eliminate κ B and ISRE regulating and controlling sequence in promoter, convert it into MHCII sample promoter.
T specificity response (intermediate value) in Fig. 4 .C57Bl/6j mice
Use 1.10
6the slow virus carrier immunity C57Bl/6j mice of TU, wherein HIV antigen presentation is subject to the driving of shown promoter.Latter 12 days of immunity, is monitored in mouse boosting cell by IFN-γ ELISPOT and replys the specific T-cells of HIV antigen.
Detailed Description Of The Invention
For determining whether the promoter existed meets following standard: in dendritic cell, high expressed is to induce maximum immunne response; With enough horizontal expressions in the cell type of other transductions, this expression is enough to eliminate for the immunne response by induction; And lack enhancer element to avoid inserting effect, the application of people's gene promoter in slow virus carrier is studied.The expression of people's promoter in dendritic cell and its hetero-organization is analyzed.Use quantitative analysis to select the high-level dendritic cell with expection to express and in a organized way in there is the promoter of higher average expression.Also select the promoter lacking enhancer sequence.
The one group of promoter meeting these standards is MHC I type promoter.MHC I type promoter display NF-Kb binding site, IFN-stimulated response element (ISRE) and SXY regulate and control the conservative of module (SXY).In MHC I type promoter, the arrangement of these elements as shown in Figure 1.
MHC II type promoter is considered to antigen presenting cell (comprising dendritic cell) specificity promoter.Equally as shown in Figure 1, although MHC II type promoter contains SXY module, it is not containing NF-Kb binding site or ISRE (Van den Helsen etc., 1998, Immunogenetics, 48:208-221).Therefore, MHC II type promoter and MHC I type promoter completely different.
Another antigen presenting cell specificity promoter dectin-2 contains IFN-stimulated response element (ISRE); But not containing SXY module (Bonkabara etc., 2001, J.Immunology, 167:6893-6900).
People's B2M (β 2m) promoter and MHC I type promoter have certain similarity, because it comprises ISRE, although be in the upstream of single NF-Kb binding site.
The sequence of multiple mammal (people) MHC I type promoter is as follows:
HLA-A2(MHC I):
attggggagtcccagccttggggattccccaactccgcagtttcttttctccctctcccaacctatgtagggtccttcttcctggatactcacgacgcggacccagttctcactcccattgggtgtcgggtttccagagaagccaatcagtgtcgtcgcggtcgcggttctaaagtccgcacgcacccaccgggactcagattctccccagacgccgagg
(SEQ ID NO:1)
HLA-B7(MHC I):
ggggaggcgcagcgttggggattccccactcccctgagtttcacttcttctcccaacttgtgtcgggtccttcttccaggatactcgtgacgcgtccccacttcccactcccattgggtattggatatctagagaagccaatcagcgtcgccgcggtcccagttctaaagtccccacgcacccacccggactcagag(SEQ ID NO:2)
HLA-Cw5(MHC I):
cactggggaggcgccgcgttgaggattctccactcccctcagtttcacttcttctcccaacctgcgtcgggtccttcttcctgaatactcatgacgcgtccccaattcccactcccattgggtgtcgggttctagagaagccaatcagcgtctccgcagtcccggtctaaagtccccagtcacccacccggactcagattctccccagacgccgag
(SEQ ID NO:3)
HLA-E(MHC I):
taagaactgctgattgctgggaaactctgcagtttcccgttcctctcgtaacctggtcatgtgtccttcttcctggatactcatgacgcagactcagttctcattcccaatgggtgtcgggtttctagagaagccaatcagcgtcgccacgactcccgactataaagtccccatccggactcaagaagttctcaggactcagagg(SEQ ID NO:4)
HLA-F(MHC I):
aggccccgaggcggtgtctggggttggaaggctcagtattgagaattccccatctccccagagtttctctttctctcccaacccgtgtcaggtccttcatcctggatactcataacgcggccccatttctcactcccattgggcgtcgcgtttctagagaagccaatcagtgtcgccgcagttcccaggttctaaagtcccacgcaccccgcgggactcatatttttcccagacgcggaggttggggtcatg(SEQ ID NO:5)
MHCI promoter (HLA-A2, HLA-B7 or HLA-E) is inserted in slow virus carrier.For comparing, the EF1 α express wide spectrum and ubiquitin (UBC) gene promoter also insert in slow virus carrier.EF1 α and ubiquitin promoter not containing the enhancer of any qualification, and do not contain SXY module, NF-Kb binding site or ISRE.On the contrary, instead, EF1 α and ubiquitin (UBC) promoter contain Sp1 and Ap1 binding site.Also prepare the carrier containing CMV promoter.Also prepare the carrier containing MHCII promoter (HLA-DR α) or β 2m promoter.In these carriers, the expression of promoters driven green fluorescent protein (GFP).
Specific expressed for finding dendritic cell, by by encapsidate plasmid and provide the plasmid co-transfection HEK 293T cell of VSV.G peplos to pack carrier, basic as Naldini etc., described in 1996, Science272:263-7.Use particle transduction HEK 293T and the BDCM cell (dendritic cell system) of different carriers subsequently.Detect the expression had in the HEK 293T cell of all carriers.
The carrier with CMV promoter, UBC promoter or EF1 α promoter is presented at expression in HEK 293T cell higher than BDCM cell (Fig. 2).But the carrier with MHCII promoter (HLA-DR α), MHCI promoter (HLA-A2, HLA-B7 or HLA-E) or β2-microglobulin promoter (β 2m) is presented at expression in BDCM cell higher than HEK 293T cell.Therefore, these promoteres all demonstrate dendritic cell specificity process LAN.
Also have evaluated the induction expressed when multiple interferon exists.Result as shown in figs 3 a andb.Interferon cannot induce MHCII promoter.But MHCI promoter can disturbed plain γ induction also can disturbed plain α induction in HEK 293T cell in whole two kinds of cell types.Similar with MHCI promoter, β 2m promoter also can disturbed element induction.The inducibility of MHCI or the β 2m promoter (promoter-SXY-GFP) of the truncate of removing ISRE and NF-Kb binding site reduces.Therefore, HLA-A2, HLA-B7, HLA-E and β 2m promoter of truncate is similar with MHCII promoter in inducibility.
Subsequently, the slow virus carrier immune mouse that wherein HIV antigen presentation drives by multiple promoter (virus, wide spectrum, MHCI, MHCII and β 2m) is used.Latter 12 days of immunity, monitors specific T-cells response in mouse boosting cell by IFN-γ ELISPOT.As shown in Figure 4, the immunity with the slow virus carrier of MHCI promoter is used to create the t cell response of most high specific.
As shown in Figure 4, use the T specificity response of MHCI promoter higher than CMV, EF1 α, ubiquitin or MHCII promoter, and be more than 3 times when using EF1 α or CMV promoter.Use the T specificity response of β 2m promoter similar with MHCI promoter.These results show, and for for using in body the response of T specificity in slow virus carrier and maximizing, MHCI promoter is unexpectedly better than EF1 α, ubiquitin, CMV or MHCII promoter.
The present invention includes the slow virus carrier comprising MHC I type (MHCI) promoter, and in host the purposes of induce immune response.
Therefore, main purpose of the present invention is a kind of slow virus carrier, described slow virus carrier comprises to come the promoter sequence of the I type mhc gene promoter of bootstrap transgene transcription, and it is in host cell, preferably optimized encoding immunogenic polypeptide in dendritic cell (DC).
slow virus carrier
Within the scope of the present invention, " slow virus carrier " refers to nonreplication vector, it is in the transgenic transduce host cells comprising cis acting slow virus RNA or DNA sequence, and needs the slow virus albumen (such as Gag, Pol and/or Env) that provides with trans forms.Slow virus carrier lacks the expression of functional Gag, Pol and Env albumen.Slow virus carrier can the form of RNA or DNA molecular exist, and this depends on generation or the developmental stage of described Retroviral Vector.
Slow virus carrier can be the form of recombinant DNA molecules, as plasmid.Slow virus carrier can be the form of lentiviral particle carrier, as slow virus and the RNA molecule in the complex of other protein.Usually, the lentiviral particle carrier corresponding to the lentiviral particle of modifying or recombinating comprises the genome be made up of two single-stranded RNA copy.These RNA sequences by transcribing acquisition from the genomic double chain DNA sequence of Insertion Into Host Cell (provirus carrier DNA), or can be obtained by the transient expression of plasmid DNA in the host cell transformed (plasmid vector DNA).
Slow virus carrier derives from slow virus, especially human immunodeficiency virus (HIV-1 or HIV-2), simian immunodeficiency virus (SIV), equine infectious encephalitis (EIAV), Caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV) and feline immunodeficiency virus (FIV), it is modified relates to pathogenic genetic determination bunch with removing and imports obtaining the useful new determinant of therapeutic effect.
This kind of carrier is separated based on cis and transacting sequence.For preparing replication-defective vector, transacting sequence (as gag, pol, tat, rev and env gene) can be deleted and use the expression cassette of encoded transgene to replace.
Effectively integrating in Unseparated Cell needs lentiviral gene group switching centre to there are two kinds of cis acting sequences with copying usually: center polypurine sequence (cPPT) and central termination sequence (CTS).This causes forming three stranded DNA structure, is called center DNA " lobe (flap) ", and it takes off bag quilt as complex before the integration to nucleopore place and expression cassette is effectively imported the nuclear signal of Unseparated Cell (as dendritic cell).
In one embodiment, the present invention includes to comprise and be called the center polypurine sequence of cPPT/CTS sequence and the slow virus carrier of central termination sequence, cPPT/CTS sequence is specifically as described in European patent application EP 2 169073.
Other sequences exist with cis form usually, as carrier proviral DNA sequence is integrated into length terminal repetition (LTR) involved in host cell gene group.As shown in Figure 1, by making LTR series jump to obtain carrier, such as suddenly change (Δ U3) (Miyoshi H etc., 1998, JVirol.72 (10): 8150-7 in the domain U3 of described LTR; Zufferey etc., 1998, J Virol 72 (12): 9873-80).
Preferably, carrier is not containing enhancer.In one embodiment, the present invention includes the slow virus carrier comprising LTR sequence, preferably there is the U3 district (Δ U3) of the sudden change removing promoter and enhancer sequence in 3 ' LTR.
Also can integrate packaging sequence Ψ (psi) to help polynucleotide sequence encapsidate to (Kessler etc., 2007, Leukemia, 21 (9): 1859-74 in carrier granular; Paschen etc., 2004, CancerImmunolImmunother 12 (6): 196-203).
In one embodiment, the present invention includes the slow virus carrier comprising slow virus packaging sequence Ψ (psi).
Preferably, other functional sequences (as transfer RNA binding site or primer binding site (PBS) or marmot posttranscriptional regulatory element (WPRE)) also can be made to be included in slow virus carrier polynucleotide sequence of the present invention to obtain the expression that in body, transgenic is more stable.
In one embodiment, the present invention includes the slow virus carrier comprising PBS.In one embodiment, the present invention includes the slow virus carrier comprising WPRE and/or IRES.
Therefore, in a preferred embodiment, slow virus carrier comprises at least one cPPT/CTS sequence, a kind of Ψ sequence, one (preferably 2 kinds) LTR sequence and expression cassette, and described expression cassette comprises the transgenic controlled by I type MHC promoter transcription.
In multiple embodiment, slow virus carrier comprises MHC I type promoter.Preferably, MHC I type promoter is HLA-A2 promoter, HLA-B7 promoter, HLA-Cw5 promoter, HLA-F or HLA-E promoter.In multiple embodiment, MHC I type promoter sequence comprise have more than 90% with promoter sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, polynucleotide sequence preferably greater than 95%, more preferably above 99% homogeny.
In multiple embodiment, compared with the carrier of transcribing control being subject to CMV promoter with transgenic sequence, the slow virus carrier comprising MHC I type promoter induces stronger CTL response in vivo for the immunogenic polypeptide of coding.
In multiple embodiment, compared with the carrier of transcribing control being subject to EF1 α promoter with transgenic sequence, the slow virus carrier comprising MHC I type promoter induces stronger CTL response in vivo for the immunogenic polypeptide of coding.Preferably, the CTL response of MHCI promoter is used than using EF1 α promoter height at least 2 times or 3 times.Within the scope of the present invention, test described in embodiment 3 can be used whether to determine carrier " compared with the carrier of transcribing control being subject to EF1 α promoter with transgenic sequence, the immunogenic polypeptide for coding induces stronger CTL response in vivo ".Also other tests that analog result is provided can be used.
In multiple embodiment, compared with the carrier of transcribing control being subject to ubiquitin promoter with transgenic sequence, the slow virus carrier comprising MHC I type promoter induces stronger CTL response in vivo for the immunogenic polypeptide of coding.Within the scope of the present invention, test described in embodiment 3 can be used whether to determine carrier " compared with the carrier of transcribing control being subject to ubiquitin promoter with transgenic sequence, the immunogenic polypeptide for coding induces stronger CTL response in vivo ".Also other tests that analog result is provided can be used.
transgenic
Within the scope of the present invention, transgenic refers to the nucleotide sequence in the slow virus carrier that stand-by slow virus carrier is transduceed, and it is not present in cell under normal circumstances.Slow virus carrier is used for this sequence to import in the cell of transduction.Term " transgenic " does not comprise those carrier sequence promoting transgenic transduction.Transgenic can be the nucleotide sequence from another organism.Or transgenic can be the nucleotide sequence from same organisms, but there are the different regulating and controlling sequences controlling it and express.Transgenic can be justice or antisense nucleic acid molecule.According to the preferred embodiment of the present invention, trangene sequence encodes immunogenic polypeptide.
Preferably, immunogenic polypeptide is virus, antibacterial or fungus.In one embodiment, immunogenic polypeptide is tumor antigen.In one embodiment, immunogenic polypeptide is allergen.
This immunogenic polypeptide preferably includes the one or more epi-positions from infectious disease cause of disease, such as, from the antigen of Gag, Pol and/or Nef albumen of HIV.
Transgenic of the present invention also codified forms several epi-positions of multi-epitope.
In certain embodiments, this kind of epi-position derives from the target antigen identified in tumor, and the mode that can obtain for the cell-mediated immune response of itself is selected.Target antigen has itemized record in the art, can select relative to several tumor types, and specifically in melanoma or cancer, comprise renal carcinoma, bladder cancer, colon cancer, pulmonary carcinoma, breast carcinoma, leukemia, myeloma and lymphoma.
mHCI promoter
The present invention includes and MHC I type (MHCI) promoter is inserted in slow virus carrier.As used herein, " MHC I type (MHCI) promoter " comprises the MHC I type promoter of natural generation or synthesis.Term " MHC I type promoter " does not comprise β 2m promoter.
The MHCI promoter of natural generation
The example of the MHCI promoter of natural generation is HLA-A2, HLA-B7, HLA-Cw5, HLA-E, HLA-F gene promoter.The MHCI promoter of these natural generations generally by the gene of coding MHC I type albumen, or is estimated as the promoter region clone of the gene of encoding such proteinaceous or copies in genome database (as: NCBI polynucleotide data base http://www.ncbi.nlm.nih.gov/guide/dna-rna).β 2m and I type MHC albumen all enter MHC (MHC).
The protein of these gene codes is found in nearly all cell type.MHCI albumen is present in leukocytic film surface usually, and now it is associated with B2M (β 2m).The effect of these protein that are associated is to CD8+T cell by the peptide submission of endogenous source.Therefore, it plays the role of a nucleus in the production process of antigen-specific immune response.Because MHC I type albumen has obtained extensively research and has described for many years, so its gene has been characterized by maturation and sequence alignment tools (as BLAST method) can be used to detect well (Altschul, S.F. etc. (1990) .Basic local alignment search tool. (Local Alignment Search basic tool) J.Mol.Biol.215 (3): 403 – 410).
MHC I type promoter also has by the ability activated by force in antigen presenting cell (comprising dendritic cell), and, in other tissue of great majority, low intensive ability is fallen.
MHC I type promoter of the present invention also can contain controlling element, as one or more Sp1 and ETs binding site.In a preferred embodiment, MHC I type promoter contains 2 Sp1 binding sites and 1 Ets binding site.In other embodiments, Ap1 and/or Ap2 site is also contained in MHC I type promoter.
Preferred promoter is people HLA-A2, HLA-B7, HLA-Cw5, HLA-E and HLA-F promoter.
The MHC I type promoter of synthesis
MHC I type promoter also can be synthesis.The MHC I type promoter of synthesizing comprises the promoter using the individual components synthesis of Protocols in Molecular Biology assembling MHC I type promoter or the promoter using Protocols in Molecular Biology to be derived by the MHC I type promoter of natural generation.
In multiple embodiment, the MHC I type promoter of synthesis comprise have more than 90% with the promoter sequence of MHC I type gene promoter (SEQ ID NO:1-5), polynucleotide sequence preferably greater than 95%, more preferably above 99% homogeny.
ISRE
Transcribing of MHC genoid is mediated by two kinds of major regulatory elements usually: IFN-stimulated response element (ISRE) and SXY module (comprising W/S, X1X2/ site α and Y/ enhancer B controlling element) (see Fig. 1).Also see Van den Elsen, Immunogenetics (1998) 48:208-211.
These regulation and control promoter elements are arranged in and extend from the region of about transcriptional start site upstream the-220 to-95 nucleotide.The tissue specificity of its mediation MHC I type gene and transcribing of cytokine induction.
The binding site of ISRE usually containing interferon regulatory factor (IRF) family member of MHC I type gene promoter.Therefore, a characteristic of MHC I type promoter is in conjunction with interferon regulatory factor (IRF) family member.This verifies by such as gel shift test.
NF-κ B binding site
There is another kind of controlling element as a rule, enhancer A (binding site containing nuclear factor κ B (NF-κ B)).Therefore, a characteristic of MHC I type promoter is in conjunction with nuclear factor κ B (NF-κ B).This verifies by such as gel shift test.
SXY module
Except ISRE, the MHC I type promoter upstream sequence motif that another group total is conservative usually, be made up of three kinds of controlling elements: S or W box, X1/CREX2 box or site α, and Y box or enhancer B, it is referred to as SXY module.This SXY module is usual and contain regulatory factor X (RFX; Be made up of RFX5, RFXB/ANK and RFXAP), cAMP response element binding protein (CREB)/activating transcription factor (ATF) and drive the multiprotein complex of the nuclear factor Y (NFY) of these gene transactivation to combine synergistically as reinforcement.Therefore, a characteristic of MHC I type promoter is in conjunction with these factors.This verifies by such as gel shift test.
On the contrary, MHC II type promoter does not show enhancer A or ISRE element (Van den Elsen, P.J etc., 1998, Immunogenetics.48:208-221).In addition, as used from bare lymphocyte symdrome (BLS; A kind of due to the severe combined immunodeficiency produced of undergoing mutation in one of RFX subunit or CIITA) shown in patient's research of carrying out of cell line of setting up, find that RFX and CIITA in MHC II type gene regulation is vital.In addition, lack function and assembling that one of CIITA or RFX subunit affects MHC reinforcement respectively, cause lacking MHC II type (Van den Elsen, the .2004 such as P.J. and MHC I type transcriptional level declines, Current Opinion in Immunology, 16:67-75).
In one embodiment, the present invention includes a kind of method, the method comprises to be inserted to instruct genetically modified expression in slow virus carrier by MHC I type promoter, described transgenic optimized encoding immunogenic polypeptide, most preferably encoding microbial antigen or therapeutic protein.The method also can comprise insertion described other nucleic acid elements arbitrarily herein, as DNA lobe sequence.
the production of lentiviral particle carrier
The invention provides the method for production lentiviral particle carrier, it does not preferably comprise promoter series but contains I type MHC promoter.Lentiviral particle carrier (or slow virus carrier granule) comprises the slow virus carrier be associated with virus protein.Carrier can be integrated or circles.
Replace the virus enhancer sequence used in prior art carrier with the safety using the gene expression of MHC I type promoters driven can improve retroviral vector:
1) it reduces the risk of the insertion mutation relevant with enhancer sequence; And
2) MHC I type promoter is activated in most (if not whole) people's cell type, thus once the immunne response for transgene product is initiated, namely remove all cells (no matter being which kind of cell type) being integrated with carrier by immune system.Therefore, after a period of time, human body can be free of any duplicate of this kind of carrier.
According to an embodiment of the method, particulate vector has in the host cell of DNA plasmid obtain in conversion.
This kind of DNA plasmid can comprise:
-bacterial origin of replication (as pUCori);
-antibiotics resistance gene (as KanR) for selecting; And more specifically:
-containing at least one genetically modified slow virus carrier relevant to MHC I type promoter transcription.
The method allows to produce according to recombinant vector granule of the present invention, comprises the following steps:
I) host cell using slow virus carrier transfection suitable;
Ii) use host cell described in the transfection of packaging plasmid carrier, described packaging plasmid carrier contains at least structure of coding retrovirus retrovirus (preferred slow virus) and the viral DNA sequences of polymerase (+/-intergrase) activity; This kind of packaging plasmid is existing in the art describes (Dull etc., 1998, J Virol, 72 (11): 8463-71; Zufferey etc., 1998, J Virol 72 (12): 9873-80).
Iii) host cell of described transfection is cultivated to make described slow virus carrier express and to be packaged into slow virus carrier granule; And
Iv) gather in the crops in the host cell of described cultivation by step I ii) expression and the slow virus carrier granule that obtains of packaging.
Based on different reason, the false type (namely increase or replace specific particle envelope albumen) of the retroviral particle prepared may be helpful.Such as, it may be favourable for having different envelope proteins to distinguish restructuring granule and native granular or other granules of recombinating.In vaccination strategies, when immune system has created for slow virus immune, false type particulate vector has more likely escaped this immune system.If the continuous injection of similar particulate vector makes patient necessary to disease generation immunity, so this is helpful especially.
For preparing the false type of retroviral particle of the present invention, one or several peplos DNA plasmid of encodes viral envelope albumen (preferred VSV-G envelope protein) also can be used to carry out transfection host cell.
Suitable host cell is preferably people's cultured cell system, such as HEK cell line.
Or can carry out in host cell for the production of the method for carrier granular, its genome has used one or more component stable conversion following: slow virus carrier DNA sequence, packaging gene and env gene.This kind of DNA sequence can be considered with provirus carrier of the present invention similar, comprises extra promoter to allow carrier sequence to transcribe and to improve particle manufacture rate.
In a preferred embodiment, also modify host cell, can produce virion in a continuous manner in the medium, whole cell does not occur swelling or dead simultaneously.About this kind of technology for the production of virion, can see Strang etc., 2005, J Virol 79 (3): 1165-71; Relander etc., 2005, MolTher 11 (3): 452-9; Stewart etc., 2009, Gene Ther, 16 (6): 805-14; With Stuart etc., 2011, Hum gene Ther (in version).
One object of the present invention forms by using the host cell of lentiviral particle vector.
Lentiviral particle carrier can comprise following element, as defined hereinabove:
-cPPT/CTS polynucleotide sequence; And
Transgenic sequence under-MHC I type promoter controls, and the one in other elements optionally mentioned above.
at cells transgene method
The present invention includes the method for express transgenic in cell (preferred Unseparated Cell).The method uses slow virus carrier of the present invention or lentiviral particle vector transduced cells under being included in the condition allowing transgene expression.
Cell is preferably mammalian cell, particularly people's cell.Particularly preferably people's Unseparated Cell.
Transgenic optimized encoding immunogenic polypeptide.The method also can comprise results or be separated described polypeptide.
Slow virus carrier or lentiviral particle carrier preferably comprise I type MHC promoter.
In one embodiment, the present invention includes a kind of method of express transgenic, comprise and make it instruct genetically modified expression MHC I type promoter insertion slow virus carrier, and use the vector transduced cells containing MHC I type promoter.
the therapeutic use of slow virus carrier
The invention still further relates to slow virus carrier of the present invention, especially lentiviral particle carrier format, for the preparation of the purposes of therapeutic composition or vaccine, described therapeutic composition or vaccine can induce or cause the immunoreation for epi-position occur or strengthen, more specifically, described epi-position is by those of transgenes encoding controlled by MHCI type promoter transcription be present in carrier.
Therefore, the invention provides the carrier as medicine or vaccine.
These carriers preferentially for treatment or the prevention of infectious disease, the disease be particularly associated with viral infection, and the disease be more particularly associated with retroviral infection (as AIDS and other immunodeficiency).
The present invention also can be used for the therapeutic scheme for tumor and cancer, is particularly useful for the scheme of immunization therapy for tumor or vaccination treatment.
Due to carrier of the present invention more specifically targeting dendritic cells to obtain cell-mediated immunne response, the CTL be particularly associated with the antigen of the transgene expression in these cells replys, its as targeting slowly or the vaccine of endogenous pathogenic microorganism (as mycobacteria or HIV virus) time it is particularly useful.
Therefore, the present invention relates to the immunogenic composition comprising defined slow virus carrier above.
Immunogenic composition of the present invention preferably comprises cPPT and the CTS sequence in carrier and carrier granular, inputs with the core of inducing or stimulate vector gene group in target cell.
During reverse transcription, cPPT and CTS sequential induction is called the formation of the three stranded DNA structure of DNA triplex, and it stimulates the core input of DNA vector sequence.Preferably, this carrier comprises the adjustment signal of transgenic and retrovirus or retrovirus sample source reverse transcription, expression and encapsidate, and said composition can induce or stimulate CTL (cytotoxic T lymphocyte) and/or CD4 to produce response to one or several epi-positions coded by the transgenic sequence existed in carrier.
Genetically modified expression is considerably improved by the mode comprising MHC I type promoter in the carrier.
Therefore, the generation or usual associated of memory CTL response can be induced, be improved to slow virus carrier of the present invention.In other words, by induction, the generation stimulating or participate in cell-mediated immune response (particularly CTL response or memory response), they can be used for the therapeutic composition for the preparation for the treatment of allergy, autoimmune disease, tumor disease or infectious disease.
Slow virus carrier of the present invention can be used for the method for Therapeutic Method and induce immune response, comprises and gives slow virus carrier to host and produce in host for genetically modified specific immune response.The cell produced in these hosts and antibody can be used as diagnostic reagent.
Slow virus carrier of the present invention directly gives patient by known route of administration, comprises systematicness, local or epidermis, intradermal, such as, in tumor route of administration.The present invention also comprises ex vivo administration, the ex vivo transduction of such as target cell, gives patient to be treated subsequently by treated cell.
In certain embodiments, immunogenic composition of the present invention directly can give patient, gives in the mode of the generation can induced, improve or participate in cell-mediated immunne response (immunne response of particularly CTL mediation) in vivo.
In another embodiment, immunogenic composition can use or repeat to give rear use by single, can produce the response that longterm memory type is cell-mediated.
A special advantage of compositions of the present invention is the cell-mediated immunne response that it can be used for causing or stimulating for multiple epi-position, described epi-position is by the transgenes encoding existed in interested nucleotide sequence or carrier or carrier granular, and it also can be used for causing or stimulating for the cell-mediated immunne response of the whole sequence product of a certain gene, such as pathogen gene maybe can be encoded the fragment of described gene of at least 8 to 15 aminoacid (preferably 9 to 12 aminoacid).
The present invention also comprises the slow virus carrier comprising nucleotide sequence, multiple repetitions (at least 2 identical sequence) of the aminoacid sequence of described nucleotide sequence coded cellular response and/or the aminoacid sequence containing at least 2 different sequences (2 epi-positions corresponding to different pathogens or tumor antigen).
Therefore, the present invention includes and can be used for compositions that is preventative and/or therapeutic vac-cination scheme, its treatment for tumor and in particular as the treatment of anticancer or anti-infectious disease.
Specifically, its can with adjuvant, other immunogenic compositions, chemotherapy or any other therapeutic treatment coupling.
Therefore, described different embodiment of the present invention, those skilled in the art it should be noted that content disclosed herein is only exemplary, and multiple other substitute, adjustment or amendment can comprise within the scope of the invention.Therefore, particular implementation shown in the invention is not restricted to herein.
Embodiment
In embodiment 1. two kinds of different cell types, the GFP of multiple promoters driven expresses.
Adopt the slow virus carrier comprising multiple promoter to build form to the HEK293T that transduces (a kind of fibroblast) and BDCM (a kind of dendritic cell like cell system) cell, and assessed the ability of described promoters driven GFP expression by flow cytometry.MFI (average fluorescent strength) in BDCM cell represents with the % of the MFI obtained in 293T cell.
Virus (CMV) and wide spectrum (UBC and EF1 α) promoter seems suppressed in BDCM, and MHC I (HLA-A2, HLA-B7 and HLA-E) and MHC II (HLA-DR α) promoter process LAN in this cell type.β 2m promoter is process LAN in this cell type also.
With every hole 1 × 10
5cell is seeded in containing in the complete medium of 10%FBS in 24 orifice plates by the density of individual cell.For each cell type, replace the mode of culture medium to 1 × 10 of 3 holes by using the virus sample dilution of 300 μ l
5individual cell is transduceed, and the cell making percentage ratio be included in 5% to 30% is transduceed.Subsequently by cell at 37 DEG C, 5%CO
2under hatch 2 hours, and every hole adds the complete medium of 1ml.Transduce latter 72 hours, trypsinization re-suspended cell, and use FACScalibur flow cytometer to adopt FL1 Air conduct measurement GFP MFI.
The induction that embodiment more than 2. kind of interferon is expressed GFP
Deposit in case at multiple interferon molecule, use the slow virus carrier comprising multiple promoter to HEK293T and BDCM cell of transduceing.With the ability that later evaluation promoters driven GFP expresses.Be also tested for MHC-I and the β 2m promoter weakening form, HLA-A2-, HLA-B7-, HLA-E-and β 2m-SXY promoter (wherein κ B and the removed promoter of ISRE regulating and controlling sequence, convert it into MHCII sample promoter).
As shown in Figure 3, in 293T or BDCM cell, interferon all cannot induce MHCII promoter.But MHCI promoter can disturbed plain γ induction also can disturbed plain α induction in 293T cell in whole two kinds of cell types.Similar with MHCI promoter, β 2m promoter also can disturbed element induction.The inducibility of MHCI or the β 2m promoter (promoter-SXY-GFP) of the truncate of removing ISRE and NF-Kb binding site reduces.Therefore, HLA-A2, HLA-B7, HLA-E and β 2m promoter of truncate is similar with MHCII promoter in inducibility.
With every hole 1 × 10
5cell is seeded in containing in the complete medium of 10%FBS in 24 orifice plates by the density of individual cell.For each cell type, replace the mode of culture medium to 1 × 10 of 12 holes by using the virus sample dilution of 300 μ l
5individual cell is transduceed, and makes percentage ratio be included in 5% to 30% cell and is transduceed.Subsequently by cell at 37 DEG C, 5%CO
2under hatch 2 hours, and in 3 holes, add the complete medium of 1ml, the 1ml complete medium containing 650U/ml (final 500U) IFN γ is added in another 3 holes, the 1ml complete medium containing 650U/ml (final 500U) IFN α is added in another 3 holes, and the 1ml complete medium added in last 3 holes containing 650U/ml (final 500U) IFN β, for each carrier transduction.Make plate at 37 DEG C, 5%CO subsequently
2under hatch.Transduce latter 72 hours, trypsinization re-suspended cell, and use FACScalibur flow cytometer to adopt FL1 Air conduct measurement GFP MFI.
T specificity response (intermediate value) in embodiment 3.C57Bl/6j mice
Use the 1x 10 that wherein HIV antigen presentation drives by multiple promoter (virus, wide spectrum, MHCI and MHCII)
6tU slow virus carrier immunity C57Bl/6j mice.Latter 12 days of immunity, monitors specific T-cells response in mouse boosting cell by IFN-γ ELISPOT.As shown in Figure 4, compared with any other slow virus carrier used, using the immune t cell response created compared with high specific of the slow virus carrier comprising MHCI promoter, is also even like this when viral promotors drives antigen presentation.
Separating Morr. cell from the mice spleen using slow virus carrier immunity.Immunity carries out ELISPOT test in latter 12 days.96 hole tissue culturing plates (Millipore Corp. (Millipore)) are spent the night with the 5 μ g/ml anti-mouse IFN γ mAb (mice IFN γ Elispot couple, BD bioscience Fa Minjin company (BD BiosciencesPharmingen)) in 50 μ l/ holes are coating at 4 DEG C.Described plate 200 μ l DPBS/ holes wash three times, and use the DPBS/10% hyclone in 200 μ l/ holes to close 2 hours at 37 DEG C.Described plate 200 μ l DPBS/ holes wash three times.With 2.5,4.1 or 5.1 × 10
5individual cells/well adds splenocyte (three multiple holes) in plate, and uses stimulator polypeptide (to antigen-specific), concanavalin A (the 5 μ g/ml of 2 μ g/ml; Original) or independent culture medium stimulate splenocyte.Plate is hatched 18 hours at 37 DEG C, uses the DPBS/0.05% polysorbas20 in 200 μ l/ holes to wash three times subsequently, and use the DPBS in 200 μ l/ holes to wash three times.For detecting, adding 2 μ g/ml anti-mouse IFN γ-biotinylated mAb (BD Fa Minjin company (BD Pharmingen)) with 50 μ l/ holes, reacting 2 hours under room temperature.Wash plate and add 100 μ l/ holes with 1:2000 streptavidin-alkali phosphatase (Roche Holding Ag (Roche)) of diluting in Dole uncle section (Dulbecco's) PBS, react 90 minutes under room temperature.After wash plate, add the BCIP/NBT solution (Sigma (Sigma)) in 60 μ l/ holes with display dot (cell of secretion IFN γ).Plate is at room temperature hatched 15-30 minute until locus coeruleus point occurs and uses tap water fully to wash and air drying 24 hours subsequently.Finally, Bioreader 2000 (Biosys Corp. (Biosys)) is used to count speckle.
Claims (14)
1. a slow virus carrier, comprises the transgenic sequence of encoding immunogenic polypeptide, and described transgenic sequence transcribes control by MHC I type promoter.
2. slow virus carrier as claimed in claim 1, the CTL response of the immunogenic polypeptide for coding that described carrier is induced in vivo is better than wherein transgenic sequence and transcribes the carrier of control by CMV promoter.
3. slow virus carrier as claimed in claim 1 or 2, described MHC I type promoter is HLA-A2 promoter, HLA-B7 promoter, HLA-Cw5 promoter, HLA-F or HLA-E promoter.
4. slow virus carrier as claimed in claim 1 or 2, described promoter sequence comprise have more than 90% with promoter sequence shown in SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, polynucleotide sequence preferably greater than 95%, more preferably above 99% homogeny.
5. the slow virus carrier according to any one of claim 1-4, described slow virus carrier comprises: the LTR of the promoter of U3 and enhancer disappearance, cPPT/CTS sequence, Ψ (psi) sequence or two slow virus LTR from slow virus.
6. the slow virus carrier according to any one of claim 1-5, described slow virus carrier is not containing enhancer.
7. the slow virus carrier according to any one of claim 1-6, described immunogenic polypeptide comprises antigen from Gag, Pol and/or Nef albumen of HIV virus or at least one tumor antigen.
8. the host cell be separated, it comprises the slow virus carrier according to any one of claim 1-7.
9. produce a method for slow virus carrier granule, said method comprising the steps of:
A) host cell using the slow virus carrier transfection according to any one of claim 1-7 suitable;
B) host cell described in the packaging plasmid carrier transfection comprising viral DNA sequences is used, at least structure of described viral DNA sequences encoding hiv reverse transcriptase and polymerase activity;
C) the described host cell of transfection is cultivated to make described slow virus carrier express and to be packaged into slow virus carrier granule; And
D) in the described host cell cultivated of results by step c) expression and the described slow virus carrier granule that obtains of packaging.
10. a slow virus carrier granule, it comprises the slow virus carrier according to any one of claim 1-7.
11. 1 kinds of slow virus carrier granules, it comprises at least following sequential element:
A) cPPT/CTS polynucleotide sequence;
B) promoter of U3 and the LTR of enhancer disappearance; And
C) by the transgenic sequence that MHC I type promoter controls.
12. slow virus carrier granules as claimed in claim 11, the sequence of described promoter comprise have more than 90% with promoter sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, polynucleotide sequence preferably greater than 95%, more preferably above 99% homogeny.
The method of 13. 1 kinds of inducing T cell responses, described method comprises and gives patient's slow virus carrier granule, described slow virus carrier granule comprises cPPT/CTS polynucleotide sequence, the LTR of the promoter of U3 and enhancer disappearance, and by the transgenic sequence that MHC I type promoter controls.
14. slow virus carrier according to any one of claim 1-7 and 11-13 or the slow virus carrier granules being used as medicine or vaccine.
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| EP12305566.7A EP2666477A1 (en) | 2012-05-23 | 2012-05-23 | Lentiviral vectors containing an MHC class I promoter |
| PCT/EP2013/059041 WO2013174630A1 (en) | 2012-05-23 | 2013-04-30 | Lentiviral vectors containing an mhc class i promoter |
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| EP3276006A1 (en) * | 2016-07-27 | 2018-01-31 | Theravectys | Lentiviral vectors for expression of hepatitis b virus (hbv) antigens |
| KR20220005208A (en) | 2020-07-06 | 2022-01-13 | 주식회사 지씨셀 | A Novel Cell for Transplantation with Reduced Immunogenicity |
| CN114107393A (en) | 2021-04-07 | 2022-03-01 | 上海劲威生物科技有限公司 | Lentiviral vector and lentiviral particle for treating hepatitis B as well as preparation methods and applications of lentiviral vector and lentiviral particle |
| KR20240073177A (en) * | 2022-11-10 | 2024-05-24 | 전남대학교산학협력단 | Pharmaceutical composition for preventing or treating glioma comprising multiple epitope peptide |
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| DE19720152A1 (en) * | 1997-05-02 | 1998-11-05 | Max Delbrueck Centrum | Retroviral vector system for gene therapy |
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| FR2777909B1 (en) | 1998-04-24 | 2002-08-02 | Pasteur Institut | USE OF TRIPLEX-STRUCTURED DNA SEQUENCES FOR THE TRANSFER OF NUCLEOTID SEQUENCES IN CELLS, RECOMBINANT VECTORS CONTAINING THESE TRIPLEX SEQUENCES |
| ES2447115T3 (en) | 1999-10-11 | 2014-03-11 | Institut Pasteur | Vectors for the preparation of immunotherapeutic compositions |
| DE60033045T2 (en) | 1999-10-12 | 2007-11-08 | Institut Pasteur | LENTIVIRAL TRIPLEX DNA, AND VECTORS AND RECOMBINANT CELLS CONTAINING LENTIVIRAL TRIPLEX DNA |
| FR2870126B1 (en) | 2004-05-17 | 2009-07-17 | Pasteur Institut | RECOMBINANT LENTIVIRAL VECTOR FOR EXPRESSION OF FLAVIVIRIDAE PROTEIN AND ITS APPLICATIONS AS VACCINE |
| US8222029B2 (en) | 2005-05-16 | 2012-07-17 | Institut Pasteur | Lentiviral vector-based vaccine |
| BRPI0813194B8 (en) | 2007-08-03 | 2021-05-25 | Centre Nat Rech Scient | kit, lentiviral vector particles, composition of plasmid vectors, chimeric hiv-1 derived antigen, vsv-g envelope protein, nucleic acid molecules, immunogenic composition and use of a lentiviral vector |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DE19720152A1 (en) * | 1997-05-02 | 1998-11-05 | Max Delbrueck Centrum | Retroviral vector system for gene therapy |
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| WO2013174630A1 (en) | 2013-11-28 |
| SG11201407732SA (en) | 2014-12-30 |
| CN104736167B (en) | 2018-07-27 |
| JP5938143B2 (en) | 2016-06-22 |
| ZA201408652B (en) | 2016-09-28 |
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| CA2873473C (en) | 2019-05-07 |
| AU2013265586B2 (en) | 2015-09-03 |
| EP2666477A1 (en) | 2013-11-27 |
| CA2873473A1 (en) | 2013-11-28 |
| EP2678032A1 (en) | 2014-01-01 |
| BR112014028944A2 (en) | 2015-09-29 |
| AU2013265586A1 (en) | 2014-12-11 |
| IL235705A (en) | 2016-10-31 |
| ES2498277T3 (en) | 2014-09-24 |
| IN2014DN10123A (en) | 2015-08-21 |
| KR101774929B1 (en) | 2017-09-19 |
| JP2015518716A (en) | 2015-07-06 |
| NZ702345A (en) | 2016-03-31 |
| BR112014028944B1 (en) | 2020-11-10 |
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